This application is related to U.S. application Ser. No. 9/314,605 filed May 19, 1999 which is incorporated by reference herein.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by any one of the patent disclosure as it appears in the U.S. Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
This invention relates to methods, apparatus, and computer program products for characterizing nucleic acid sequences, and more particularly to methods, apparatus and computer program products for determining quantities of nucleic acid sequences in samples.
Quantitative nucleic sequence analysis plays an increasingly important role in the fields of biological and medical research. For example, quantitative gene analysis has been used to determine the genome quantity of a particular gene, as in the case of the human HER-2 oncogene, which is amplified in approximately 30% of human breast cancers. Gene and genome quantitation have also been used in determining and monitoring the levels of human immunodeficiency virus (HIV) in a patient throughout the different phases of the HIV infection and disease. It has been suggested that higher levels of circulating HIV and failure to effectively control virus replication after infection may be associated with a negative disease prognosis. Accordingly, an accurate determination of nucleic acid levels early in an infection may serve as a useful tool in diagnosing illness, while the ability to correctly monitor the changing levels of viral nucleic acid in one patient throughout the course of an illness may provide clinicians with critical information regarding the effectiveness of treatment and progression of disease.
Several methods have been described for the quantitative analysis of nucleic acid sequences. The polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) permit 30 the analysis of small starting quantities of nucleic acid (e.g., as little as one cell equivalent). Early methods for quantitation involved measuring PCR product at the end of temperature thermal cycling and relating this level to the starting DNA concentration. Unfortunately, the absolute amount of product generated does not always bear a consistent relationship to the amount of target sequence present at the initiation of the reaction, particularly for clinical samples. Such an endpoint analysis reveals the presence or absence of starting nucleic acid, but generally does not provide an accurate measure of the number of DNA targets. Both the kinetics and efficiency of amplification of a target sequence are dependent on the starting quantity of that sequence, and on the sequence match of the primers and target template, and may also be affected by inhibitors present in the sample. Consequently, endpoint measurements have very poor reproducibility.
Another method, quantitative competitive PCR (QC-PCR), has been developed and used widely for PCR quantitation. QC-PCR relies on the inclusion of a known amount of an internal control competitor in each reaction mixture. To obtain relative quantitation, the unknown target PCR product is compared with the known competitor PCR product, usually via gel electrophoresis. The relative amount of target-specific and competitor DNA is measured, and this ratio is used to calculate the starting number of target templates. The larger the ratio of target specific product to competitor specific product, the higher the starting DNA concentration. Success of a QC-PCR assay relies on the development of an internal control that amplifies with the same efficiency as the target molecule. However, the design of the competitor and the validation of amplification efficiencies require much effort. In the QC-PCR method of RNA quantitation, a competitive RNA template matched to the target sequence of interest, but different from it by virtue of an introduced internal deletion, is used in a competitive titration of the reverse transcription and PCR steps, providing stringent internal control. Increasing amounts of known copy numbers of competitive template are added to replication portions of the test sample, and quantitation is based on determination of the relative (not absolute) amounts of the differently sized amplified products derived from the wild-type and competitive templates, after electrophoretic separation.
In addition to requiring time-consuming and burdensome downstream processing such as hybridization or gel electrophoresis, these assays have limited sensitivity to a range of target nucleic acid concentrations. For example, in competitor assays, the sensitivity to template concentration differences may be compromised when either the target or added competitor DNA is greatly in excess of the other. The dynamic range of the assays that measure the amount of end product can also be limited in that the chosen number of cycles of some reactions may have reached a plateau level of product prior to other reactions. Differences in starting template levels in these reactions may therefore not be well reflected. Furthermore, small differences in the measured amount of product may result in widely varying estimates of the starting template concentration, leading to inaccuracies due to variable reaction conditions, variations in sampling, or the presence of inhibitors.
To reduce the amount of post-amplification analysis required to determine a starting nucleic acid quantity in a sample, additional methods have been developed to measure nucleic acid amplification in real-time. These methods generally take advantage of fluorescent labels (e.g., fluorescent dyes) that indicate the amount of nucleic acid being amplified, and utilize the relationship between the number of cycles required to achieve a chosen level of fluorescence signal and the concentration of amplifiable targets present at the initiation of the PCR process. For example, European Patent Application No. 94112728 (Publication number EP/0640828) describes a quantitative assay for an amplifiable nucleic acid target sequence which correlates the number of thermal cycles required to reach a certain concentration of target sequence to the amount of target DNA present at the beginning of the PCR process. In this assay system, a set of reaction mixtures are prepared for amplification, with one preparation including an unknown concentration of target sequence in a test sample and others containing known concentrations (standards) of the sequence. The reaction mixtures also contain a fluorescent dye that fluoresces when bound to double-stranded DNA.
The reaction mixtures are thermally cycled in separate reaction vessels for a number of cycles to achieve a sufficient amplification of the targets. The fluorescence emitted from the reaction mixtures is monitored in real-time as the amplification reactions occur, and the number of cycles necessary for each reaction mixture to fluoresce to an arbitrary cutoff level (arbitrary fluorescent value, or AFV) is determined. The AFV is chosen to be in a region of the amplification curves that is parallel among the different standards (e.g., from 0.1 to 0.5 times the maximum fluorescence value obtained by the standard using the highest initial known target nucleic acid concentration). The number of cycles necessary for each of the standards to reach the AFV is determined, and a regression line is fitted to the data that relates the initial target nucleic acid amount to the number of cycles (i.e., the threshold cycle number) needed to reach the AFV. To determine the unknown starting quantity of the target nucleic acid sequence in the sample, the number of cycles needed to reach the AFV is determined for the sample. This threshold cycle number (which can be fractional) is entered into the equation of the fitted regression line and the equation returns a value that is the initial amount of the target nucleic acid sequence in the sample.
The primary disadvantage of this method for determining an unknown starting quantity of a target nucleic acid sequence in a sample is that differences in background signal, noise, or reaction efficiency between the reaction mixtures being amplified in different reaction vessels may bias the calculation of the threshold cycle numbers. Consequently, several of the data points used to generate the regression line may deviate significantly from linearity, resulting in inaccurate quantitation of the unknown starting quantity of the target nucleic acid sequence in the sample. Small differences in the selection of threshold cycle numbers used in quantitation algorithms may have a substantial effect on the ultimate accuracy of quantitation. Thus, there remains a need to provide an objective and automatic method of selecting threshold values that will allow users of amplification methods to determine the initial concentrations of target nucleic acid sequences more accurately and reliably than present methods.
It is therefore an object of the present invention to provide improved methods, apparatus, and computer program products for determining a threshold value in a nucleic acid amplification reaction. The threshold value may be a threshold cycle number in a thermal cycling amplification reaction, or the threshold value may be a time value (e.g., an elapsed time of amplification) in an isothermal nucleic acid amplification reaction.
It is another object of the present invention to provide improved methods, apparatus, and computer program products for determining quantities of nucleic acid sequences in samples.
According to a first embodiment, the invention provides a method for determining a threshold cycle number (which may be fractional) in a nucleic acid amplification reaction. The method includes the step of amplifying a nucleic acid sequence in a sample by subjecting the sample to thermal cycling. Suitable thermal cycling methods include, but are not limited to, the Polymerase Chain Reaction (PCR); Reverse Transcriptase PCR (RT-PCR); Ligase Chain Reaction (LCR); and transcription-based amplification. The method also includes the step of measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of the nucleic acid sequence in the sample. Preferably, the sample contains a fluorescent indicator, and the signal is a fluorescent signal whose intensity is proportional to the quantity of the nucleic acid sequence in the sample. The method also includes the steps of deriving a growth curve (e.g., signal intensity as a function of cycle number) from the measurements of the signal, calculating a derivative of the growth curve, identifying a characteristic of the derivative, and determining a cycle number associated with the characteristic of the derivative.
The step of calculating a derivative of the growth curve referably comprises calculating second derivative values of the growth curve at a number of different cycles in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the cycle number associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the threshold cycle number as the location, in cycles, of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the threshold cycle number may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
The present invention also provides an apparatus for monitoring a nucleic acid amplification reaction in real-time and for determining a threshold cycle number in the reaction. The apparatus comprises a detection mechanism for measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of a nucleic acid sequence being amplified in the reaction. The apparatus also includes a controller (e.g., a computer or processor) in communication with the detection mechanism. The controller is programmed to perform the steps of deriving a growth curve from the measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a cycle number associated with the characteristic of the derivative. The step of calculating a derivative of the growth curve preferably comprises calculating second derivative values of the growth curve at a number of different cycles in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the cycle number associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the threshold cycle number as the location, in cycles, of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the threshold cycle number may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
According to another aspect, the invention provides a computer program product readable by a machine having at least one detection mechanism operatively coupled thereto for detecting and measuring at a plurality of different times during a nucleic acid amplification reaction at least one signal whose intensity is related to a quantity of a nucleic acid sequence being amplified in the reaction. The computer program product embodies a program of instructions executable by the machine to perform the steps of deriving a growth curve from measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a cycle number associated with the characteristic of the derivative. The step of calculating a derivative of the growth curve preferably comprises calculating second derivative values of the growth curve at a number of different cycles in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the cycle number associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the threshold cycle number as the location, in cycles, of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the threshold cycle number may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
According to a second embodiment, the invention provides a method for determining a threshold time value in a nucleic acid amplification reaction. The method is particularly useful for determining a threshold time value (e.g., an elapsed time of amplification required to reach a threshold level) in isothermal nucleic acid amplification reactions. Suitable isothermal amplification methods include, but are not limited to, Rolling Circle Amplification; Strand Displacement Amplification (SDA); Q-beta replicase; Nucleic Acid-Based Sequence Amplification (NASBA); and Self-Sustained Sequence Replication (3SR). The method includes the steps of amplifying a nucleic acid sequence in a sample and measuring, at a plurality of different times during the amplification reaction, at least one signal whose intensity is related to the quantity of the nucleic acid sequence in the sample. Preferably, the sample contains a fluorescent indicator, and the signal is a fluorescent signal whose intensity is proportional to the quantity of the nucleic acid sequence in the sample. The method also includes the steps of deriving a growth curve (e.g., signal intensity as a function of elapsed time of amplification) from the measurements of the signal, calculating a derivative of the growth curve, identifying a characteristic of the derivative, and determining a time value associated with the characteristic of the derivative.
The step of calculating the derivative of the growth curve preferably comprises calculating second derivative values of the growth curve at a number of different times in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the time value associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the time value as the location of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the time value may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
The present invention also provides an apparatus for monitoring a nucleic acid amplification reaction in realtime and for determining a threshold time value in the reaction. The apparatus comprises a detection mechanism for measuring, at a plurality of different times during the. amplification reaction, at least one signal whose intensity is related to the quantity of a nucleic acid sequence being amplified in the reaction. The apparatus also includes a controller (e.g., a computer or processor) in communication with the detection mechanism. The controller is programmed to perform the steps of deriving a growth curve from the measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a time value associated with the characteristic of the derivative. The step of calculating a derivative of the growth curve preferably comprises calculating second derivative values of the growth curve at a number of different times in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the time value associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the threshold time value as the location of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the threshold time value may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
According to another aspect, the invention provides a computer program product readable by a machine having at least one detection mechanism operatively coupled thereto for detecting and measuring at a plurality of different times during a nucleic acid amplification reaction at least one signal whose intensity is related to a quantity of a nucleic acid sequence being amplified in the reaction. The computer program product embodies a program of instructions executable by the machine to perform the steps of deriving a growth curve from measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a time value associated with the characteristic of the derivative. The step of calculating a derivative of the growth curve preferably comprises calculating second derivative values of the growth curve at a number of different times in the reaction to yield a plurality of second derivative data points. The characteristic of the derivative is preferably a positive peak of the second derivative, and the step of determining the time value associated with the positive peak preferably comprises fitting a second order curve to the second derivative data points and calculating the threshold time value as the location of a peak of the second order curve. Alternatively, the characteristic of the derivative used to determine the threshold time value may comprise a negative peak of the second derivative, a zero crossing of the second derivative, or a positive peak of the first derivative.
According to another embodiment, the invention provides a method for determining an unknown starting quantity of a target nucleic acid sequence in a test sample. The method comprises the steps of amplifying the unknown starting quantity of the target nucleic acid sequence in the test sample and amplifying a plurality of known starting quantities of a calibration nucleic acid sequence in respective calibration samples (i.e., standards). The method also includes the step of determining a respective threshold value (e.g., a threshold cycle number or time value) for each of the known starting quantities of the calibration nucleic acid sequence in the calibration samples and for the target nucleic acid sequence in the test sample. The threshold value is determined for each nucleic acid sequence in a respective sample by measuring, at a plurality of different times during amplification, at least one signal whose intensity is related to the quantity of the nucleic acid sequence being amplified; deriving a growth curve from the measurements of the signal; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining a threshold value associated with the characteristic of the derivative.
The method also includes the steps of deriving a calibration curve from the threshold values determined for the known starting quantities of the nucleic acid sequence in the calibration samples and determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the target sequence. One advantage of this method is that highly reproducible threshold values are obtained even when there is significant variation (e.g., in terms of timing, optics, or noise due to other sources) between the reaction sites at which the various test and calibration samples are amplified. The threshold value for each target nucleic acid sequence being amplified in a particular reaction is based solely on the data from that reaction, not from all of the reactions in a batch. This is important because a single discrepant reaction in the batch will not bias the calculation of threshold values for target nucleic acid sequences at other reaction sites.
The present invention also provides an apparatus for determining an unknown starting quantity of a target nucleic acid sequence in a test sample. The apparatus comprises means for amplifying the unknown starting quantity of the target nucleic acid sequence in the test sample and for amplifying a plurality of known starting quantities of a calibration nucleic acid sequence in respective calibration samples. The apparatus also includes at least one detection mechanism for measuring, at a plurality of different times during amplification of the nucleic acid sequences, signals indicative of the quantities of the nucleic acid sequences being amplified in the test and calibration samples. The apparatus further includes at least one controller (e.g., computer or processor) in communication with the detection mechanism. The controller is programmed to determine a respective threshold value for each of the known starting quantities of the calibration nucleic acid sequence in the calibration samples and for the target nucleic acid sequence in the test sample. Each threshold value is determined for a nucleic acid sequence in a respective sample by deriving a growth curve for the nucleic acid sequence from the measured signals; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining the threshold value associated with the characteristic of the derivative. The controller is also programmed to derive a calibration curve from the threshold values determined for the known starting quantities of the nucleic acid sequence in the calibration samples and to determine the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the target sequence.
According to another aspect, the invention provides a computer program product readable by a machine having at least one detection mechanism operatively coupled thereto for detecting and measuring signals indicative of the quantities of a target nucleic acid sequence being amplified in a test sample, containing an unknown starting quantity of the target nucleic acid sequence therein, and of a calibration nucleic acid sequence being amplified in a plurality of calibration samples, containing respective known starting quantities of the calibration nucleic acid sequence therein. The computer program product embodies a program of instructions executable by the machine to perform the step of determining a respective threshold value for the target nucleic acid sequence in the test sample and for each of the known starting quantities of the calibration nucleic acid sequence in the calibration samples. Each threshold value is determined for a nucleic acid sequence in a respective sample by deriving a growth curve for the nucleic acid sequence from the measured signals; calculating a derivative of the growth curve; identifying a characteristic of the derivative; and determining the threshold value associated with the characteristic of the derivative. The computer program product further embodies a program of instructions executable by the machine to perform the step of deriving a calibration curve from the threshold values determined for the known starting quantities of the nucleic acid sequence in the calibration samples and determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the target sequence.
According to another embodiment, the invention provides a method for determining an unknown starting quantity of a target nucleic acid sequence in a test sample using quantitative internal controls. The method includes the step of amplifying the target nucleic acid sequence and a first internal control in a first nucleic acid amplification reaction. The first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence in the test sample. The method also includes the step of amplifying a first standard and a second internal control in a second nucleic acid amplification reaction. The first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and the second internal control comprises a known quantity of the second nucleic acid sequence. The method further includes the step of amplifying a second standard and a third internal control in a third nucleic acid amplification reaction. The second standard comprises a second known starting quantity of the calibration nucleic acid sequence, and the third internal control comprises a known quantity of the second nucleic acid sequence. The starting quantity of the second nucleic acid sequence is substantially equal in each of the amplification reactions.
Next, a respective threshold value is determined for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample. The threshold value determined for the target nucleic acid sequence in the test sample is then normalized to the threshold value determined for the first internal control. In addition, the threshold values determined for the first and second standards are normalized to the threshold values determined for the second and third internal controls, respectively. The method also includes the steps of deriving a calibration curve from the known starting quantities and the normalized threshold values of the first and second standards and determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target sequence. The normalization of the threshold values to an internal control corrects for factors affecting the different reactions (e.g., the presence of inhibitors or unstable enzymes in the reaction). The threshold values are normalized for any such effects to provide greater accuracy in the calibration curve and in the quantitation of the unknown quantity of the target sequence in the test sample.
The invention also provides an apparatus for determining an unknown starting quantity of a target nucleic acid sequence in a test sample. The apparatus includes at least one detection mechanism for measuring (1) signals indicative of the respective quantities of the target nucleic acid sequence and of a first internal control being amplified in a first nucleic acid amplification reaction, wherein the first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence; (2) signals indicative of the respective quantities of a first standard and of a second internal control being amplified in a second nucleic acid amplification reaction, wherein the first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and wherein the second internal control comprises the second nucleic acid sequence; and (3) signals indicative of the respective quantities of a second standard and of a third internal control being amplified in a third nucleic acid amplification reaction, wherein the second standard comprises a second known starting quantity of the calibration nucleic acid sequence, the third internal control comprises the second nucleic acid sequence, and the starting quantity of the second nucleic acid sequence is substantially equal in each of the amplification reactions. The apparatus also includes at least one controller (e.g., computer or processor) in communication with the detection mechanism. The controller is programmed to (a) determine from the measured signals a respective threshold value for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample; (b) normalize the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control; (c) normalize the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively; (d) derive a calibration curve from the known starting quantities and the normalized threshold values of the first and second standards; and (e) determine the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target nucleic acid sequence.
According to another aspect, the invention provides a computer program product readable by a machine having at least one detection mechanism for measuring (1) signals indicative of the respective quantities of a target nucleic acid sequence in a test sample and of a first internal control being amplified in a first nucleic acid amplification reaction, wherein the first internal control comprises a second nucleic acid sequence different than the target nucleic acid sequence in the test sample; (2) signals indicative of the respective quantities of a first standard and of a second internal control being amplified in a second nucleic acid amplification reaction, wherein the first standard comprises a first known starting quantity of a calibration nucleic acid sequence different than the second nucleic acid sequence, and wherein the second internal control comprises the second nucleic acid sequence; and (3) signals indicative of the respective quantities of a second standard and of a third internal control being amplified in a third nucleic acid amplification reaction, wherein the second standard comprises a second known starting quantity of the calibration nucleic acid sequence, the third internal control comprises the second nucleic acid sequence, and the starting quantity of the second nucleic acid sequence is substantially the same in each of the amplification reactions. The computer program product embodies a program of instructions executable by the machine to perform the steps of (a) determining a respective threshold value for each of the standards, each of the internal controls, and the target nucleic acid sequence in the test sample; (b) normalizing the threshold value determined for the target nucleic acid sequence in the test sample to the threshold value determined for the first internal control; (c) normalizing the threshold values determined for the first and second standards to the threshold values determined for the second and third internal controls, respectively; (d) deriving a calibration curve from the known starting quantities and the normalized threshold values of the first and second standards; and (e) determining the starting quantity of the target nucleic acid sequence in the test sample using the calibration curve and the normalized threshold value determined for the target sequence.
According to another embodiment, the invention provides a method for determining an unknown starting quantity of a first target nucleic acid sequence in a test sample by amplifying the first nucleic acid sequence together with a plurality of standards in the same reaction vessel. The method includes the steps of amplifying in one reaction vessel the first nucleic acid sequence, a first standard, and a second standard. The first standard comprises a known starting quantity of a second nucleic acid sequence different than the first nucleic acid sequence, and the second standard comprises a different known starting quantity of a third nucleic acid sequence different than the first and second sequences. The method also includes the steps of determining a respective threshold value (e.g., a threshold cycle number or elapsed time of amplification) for the first standard, the second standard, and the first nucleic acid sequence. A calibration curve is derived from the known starting quantities and from the threshold values determined for the first and second standards. The method further includes the step of determining the starting quantity of the first nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the first nucleic acid sequence. One advantage of this method is that a calibration curve is developed based only on the reaction in which the unknown quantity of the target nucleic acid sequence is being amplified. Consequently, the method reduces problems arising from the variability between reactions occurring in different reaction vessels. Another advantage of the method is that it reduces the number of reaction sites and the amount of expensive reagents required to perform an assay.
The invention also provides an apparatus for determining an unknown starting quantity of a first nucleic acid sequence in a test sample. The apparatus comprises a detection mechanism for detecting and measuring signals indicative of the respective quantities of the first nucleic acid sequence, a first standard, and a second standard being amplified in a reaction vessel. The first standard comprises a known starting quantity of a second nucleic acid sequence different than the first nucleic acid sequence, and the second standard comprises a known starting quantity of a third nucleic acid sequence different than the first and second sequences. The apparatus also includes at least one controller in communication with the detection mechanism. The controller is programmed to perform the steps of (a) determining a respective threshold value for the first standard, second standard, and first nucleic acid sequence; (b) deriving a calibration curve from the known starting quantities and from the threshold values determined for the first and second standards; and (c) determining the starting quantity of the first nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the first nucleic acid sequence.
According to another aspect, the invention provides a computer program product readable by a machine having at least one detection mechanism operatively coupled thereto for detecting and measuring signals indicative of the respective quantities of a first nucleic acid sequence, a first standard, and a second standard being amplified in a reaction vessel, wherein the first standard comprises a known starting quantity of a second nucleic acid sequence different than the first nucleic acid sequence, and wherein the second standard comprises a known starting quantity of a third nucleic acid sequence different than the first and second sequences. The computer program product embodies a program of instructions executable by the machine to perform the steps of (a) determining respective threshold values for the first nucleic acid sequence, first standard, and second standard (b) deriving a calibration curve from the known starting quantities and from the threshold values determined for the first and second standards; and (c) determining the starting quantity of the first nucleic acid sequence in the test sample using the calibration curve and the threshold value determined for the first nucleic acid sequence.
A more complete understanding of the methods, apparatus, and computer program products of the present invention may be gained upon consideration of the following description and accompanying drawings.